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T. Nagasaki, J. Zhao; Time-Lapse Observation of Cell Movement in the Conjunctival Epithelium Near the Limbus . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3774.
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Purpose: To determine movements of epithelial cells in the bulbar conjunctiva near the limbus and compare them with those of corneal epithelial cells, and also to gain insight into the homeostasis of conjunctival epithelium. Methods: The mouse line that was engineered to expresses GFP ubiquitously was used. Distribution of GFP positive cells was determined with a fixed whole mount specimen and also with serial frozen cross-sections of a conjunctival sac. For an in vivo observation of GFP positive cells, the mouse was anesthetized, the eye proptosed, and the GFP fluorescence in the inferior temporal quadrant of the limbus and the conjunctiva was imaged with a fluorescence microscope. Since only a limited area of a microscopic field was in focus due to curvature of the eye, five to ten overlapping fields were recorded to document an area consisting of about 0.5 mm to both sides of the limbus and 2-3 mm along the length of the limbus. GFP patterns were recorded 3-4 times per week for several weeks and the movement of GFP positive cells was analyzed from time-lapse sequences. Results: Similar to the corneal epithelium, only some of the conjunctival epithelial cells were highly positive with GFP, and this allowed relocation and tracking of the same cells over time. However, GFP positive cells in the conjunctiva did not exhibit striped patterns that were common in the corneal epithelium. Some GFP positive cells in the conjunctiva were more brighter than those in the corneal epithelium, with a size of bright cell clusters ranging from several cells to several dozens of cells. Histology of an entire limbal circumference revealed no preferential localization of GFP positive cells, nor was there any correlation between the presence of GFP positive corneal epithelial cells and that of GFP positive conjunctival cells. Time-lapse analyses revealed that most GFP positive cell clusters of the conjunctiva were stationary for a week or more. Some clusters gradually changed their shape but no directional movement was obvious. Occasionally there was a dramatic change of a cluster shape over a two-day period, perhaps representing an accidental physical insult on the conjunctiva. Some clusters persisted for 4 weeks or more without changing their location or cell distribution patterns, such that a unique geometric shape of a GFP positive cell cluster was maintained for the entire duration. This was true in both cells at the immediate vicinity of the limbus and those at more than 0.5 mm away from it. Conclusions: The epithelial cells of the bulbar conjunctiva near the limbus are largely immobile in a lateral direction in contrast to the corneal epithelial cells which exhibit a constant centripetal movement.
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